The broad objectives of this R21 Stem Cells and Cancer proposal are to 1) to establish the identity and characterize the tumorigenic potential of the MM-CSC and 2) to determine whether N-cadherin interaction between MM-CSC and stroma is required to maintain the proliferative quiescence of the MM-CSC. This work will open a gateway to study the signaling pathways governing the proliferation and differentiation of the MM-CSC, and once those are identified, new drugs can be designed to prevent the interaction between the differentiation-inducing factors and the MM-CSC, thus creating a first MM-CSC targeted therapeutic agent capable of preventing tumor initiation and relapse. Rationale: Strong evidence in support of the cancer stem cell (CSC) theory has been steadily accumulating over the last decade. In addition to tumorigenic potential, a CSC possesses characteristics of normal stem cells including quiescence and self-renewal potential. Upon receiving proliferation signal(s) from the microenvironment, a CSC switches its program from quiescence to differentiation and proliferation, initiating tumor growth. While patients with solid tumors may benefit from the surgical removal of their malignant outgrowth, hopefully along with drug-resistant tumor-initiating CSC, the surgical option is not available for the patients suffering from hematological malignancies. Such patients have a high rate of relapse due to inability of the currently used therapies to target successfully CSC. Thus, determining which characteristics of the CSC can be therapeutically exploited is of utmost importance. Multiple myeloma (MM) is an incurable malignancy of the bone marrow (BM). Nearly all MM patients relapse indicating that the drug-resistant MM cancer stem cell (MM-CSC), the identity of which is currently a subject of investigation, escapes these therapies. Hypothesis: MM-CSCs are found in a specialized microenvironment niche which maintains these cells in a non-proliferative state. Altering the microenvironment in favor of differentiation leads to tumor growth. Specific Aims: Aim 1: To define the phenotype of the MM-CSC. Aim 2: To determine whether N-cadherin interactions between stroma and MM-CSC are required to maintain the MM-CSC niche. Overall approach: We have recently developed a three-dimensional (3-D) ex vivo tissue culture model recapitulating the architecture and composition of the human BM. This model makes proliferating and non-proliferating compartments of the MM BM accessible for further analysis where the identity, clonality, self-renewal, and generative and tumorigenic potential of MM stem cells can be established. To establish the identity of the MM-CSC and to define its niche this proposal will combine the use of the 3-D tissue culture system with xenograft mouse models.